Compositions and Methods for Diagnosing and Treating Cancer

ABSTRACT

An isolated antibody that specifically binds to an extracellular domain of human DDR2 and has a therapeutic effect on a solid tumor is described. Also described is a method of treating cancer, the method comprising administering to a patient having a solid tumor an antibody of the present disclosure in a therapeutically effective amount.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional applicationSer. No. 11/951,039, filed Dec. 5, 2007, which claims the benefit ofU.S. Provisional Application 60/872,792, filed Dec. 5, 2006, which isherein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of oncology and providesnovel compositions and methods for diagnosing and treating cancer. Thepresent invention provides antibodies against a cancer stem cell markerfor the diagnosis and treatment of solid tumors.

2. Background Art

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich—breast, lung, colorectal, and prostate—account for over half ofall new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

Breast cancer is the most common cancer in women, with an estimate 12%of women at risk of developing the disease during their lifetime.Although mortality rates have decreased due to earlier detection andimproved treatments, breast cancer remains a leading cause of death inmiddle-aged women, and metastatic breast cancer is still an incurabledisease. On presentation, most patients with metastatic breast cancerhave only one or two organ systems affected, but as the diseaseprogresses, multiple sites usually become involved. The most commonsites of metastatic involvement are locoregional recurrences in the skinand soft tissues of the chest wall, as well as in axilla andsupraclavicular areas. The most common site for distant metastasis isthe bone (30-40% of distant metastasis), followed by the lungs andliver. And although only approximately 1-5% of women with newlydiagnosed breast cancer have distant metastasis at the time ofdiagnosis, approximately 50% of patients with local disease eventuallyrelapse with metastasis within five years. At present the mediansurvival from the manifestation of distant metastases is about threeyears.

Current methods of diagnosing and staging breast cancer include thetumor-node-metastasis (TNM) system that relies on tumor size, tumorpresence in lymph nodes, and the presence of distant metastases(American Joint Committee on Cancer: AJCC Cancer Staging Manual.Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed., 1997, pp171-180; Harris, J R: “Staging of breast carcinoma” in Harris, J. R.,Hellman, S., Henderson, I. C., Kinne D. W. (eds.): Breast Diseases.Philadelphia, Lippincott, 1991). These parameters are used to provide aprognosis and select an appropriate therapy. The morphologic appearanceof the tumor can also be assessed but because tumors with similarhistopathologic appearance can exhibit significant clinical variability,this approach has serious limitations. Finally assays for cell surfacemarkers can be used to divide certain tumors types into subclasses. Forexample, one factor considered in the prognosis and treatment of breastcancer is the presence of the estrogen receptor (ER) as ER-positivebreast cancers typically respond more readily to hormonal therapies suchas tamoxifen or aromatase inhibitors than ER-negative tumors. Yet theseanalyses, though useful, are only partially predictive of the clinicalbehavior of breast tumors, and there is much phenotypic diversitypresent in breast cancers that current diagnostic tools fail to detectand current therapies fail to treat.

Prostate cancer is the most common cancer in men in the developed world,representing an estimated 33% of all new cancer cases in the U.S., andis the second most frequent cause of death (Jemal et al., 2003, CACancer J. Clin. 53:5-26). Since the introduction of the prostatespecific antigen (PSA) blood test, early detection of prostate cancerhas dramatically improved survival rates; the five year survival ratefor patients with local and regional stage prostate cancers at the timeof diagnosis is nearing 100%. Yet more than 50% of patients willeventually develop locally advanced or metastatic disease(Muthuramalingam et al., 2004, Clin. Oncol. 16:505-16).

Currently radical prostatectomy and radiation therapy provide curativetreatment for the majority of localized prostate tumors. However,therapeutic options are very limited for advanced cases. For metastaticdisease, androgen ablation with luteinising hormone-releasing hormone(LHRH) agonist alone or in combination with anti-androgens is thestandard treatment. Yet despite maximal androgen blockage, the diseasenearly always progresses with the majority developingandrogen-independent disease. At present there is no uniformly acceptedtreatment for hormone refractory prostate cancer, and chemotherapeuticregimes are commonly used (Muthuramalingam et al., 2004, Clin. Oncol.16:505-16; Trojan et al., 2005, Anticancer Res. 25:551-61).

Colorectal cancer is the third most common cancer and the fourth mostfrequent cause of cancer deaths worldwide (Weitz et al., 2005, Lancet365:153-65). Approximately 5-10% of all colorectal cancers arehereditary with one of the main forms being familial adenomatouspolyposis (FAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Colorectal carcinomas invade locally bycircumferential growth and elsewhere by lymphatic, hematogenous,transperitoneal, and perineural spread. The most common site ofextralymphatic involvement is the liver, with the lungs the mostfrequently affected extra-abdominal organ. Other sites of hematogenousspread include the bones, kidneys, adrenal glands, and brain.

The current staging system for colorectal cancer is based on the degreeof tumor penetration through the bowel wall and the presence or absenceof nodal involvement. This staging system is defined by three majorDuke's classifications: Duke's A disease is confined to submucosa layersof colon or rectum; Duke's B disease has tumors that invade through themuscularis propria and may penetrate the wall of the colon or rectum;and Duke's C disease includes any degree of bowel wall invasion withregional lymph node metastasis. While surgical resection is highlyeffective for early stage colorectal cancers, providing cure rates of95% in Duke's A patients, the rate is reduced to 75% in Duke's Bpatients and the presence of positive lymph node in Duke's C diseasepredicts a 60% likelihood of recurrence within five years. Treatment ofDuke's C patients with a post surgical course of chemotherapy reducesthe recurrence rate to 40%-50% and is now the standard of care for thesepatients.

Lung cancer is the most common cancer worldwide, the third most commonlydiagnosed cancer in the United States, and by far the most frequentcause of cancer deaths (Spiro et al., 2002, Am. J. Respir. Crit. CareMed. 166:1166-96; Jemal et al., 2003, CA Cancer J. Clin. 53:5-26).Cigarette smoking is believed responsible for an estimated 87% of alllung cancers making it the most deadly preventable disease. Lung canceris divided into two major types that account for over 90% of all lungcancers: small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC). SCLC accounts for 15-20% of cases and is characterized by itsorigin in large central airways and histological composition of sheetsof small cells with little cytoplasm. SCLC is more aggressive thanNSCLC, growing rapidly and metastasizing early. NSCLC accounts for80-85% of all cases and is further divided into three major subtypesbased on histology: adenocarcinoma, squamous cell carcinoma (epidermoidcarcinoma), and large cell undifferentiated carcinoma.

Lung cancer typically presents late in its course, and thus has a mediansurvival of only 6-12 months after diagnosis and an overall 5 yearsurvival rate of only 5-10%. Although surgery offers the best chance ofa cure, only a small fraction of lung cancer patients are eligible withthe majority relying on chemotherapy and radiotherapy. Despite attemptsto manipulate the timing and dose intensity of these therapies, survivalrates have increased little over the last 15 years (Spiro et al., 2002,Am. J. Respir. Crit. Care Med. 166:1166-96).

These four cancers, as well as many others, present as solid tumors thatare composed of heterogeneous cell populations. For example, breastcancers are a mixture of cancer cells and normal cells, includingmesenchymal (stromal) cells, inflammatory cells, and endothelial cells.Several models of cancer provide different explanations for the presenceof this heterogeneity. One model, the classic model of cancer, holdsthat phenotypically distinct cancer cell populations all have thecapacity to proliferate and give rise to a new tumor. In the classicalmodel, tumor cell heterogeneity results from environmental factors aswell as ongoing mutations within cancer cells resulting in a diversepopulation of tumorigenic cells. This model rests on the idea that allpopulations of tumor cells have some degree of tumorigenic potential.(Pandis et al., 1998, Genes, Chromosomes & Cancer 12:122-129; Kuukasjrviet al., 1997, Cancer Res. 57:1597-1604; Bonsing et al., 1993, Cancer71:382-391; Bonsing et al., 2000, Genes Chromosomes & Cancer 82:173-183; Beerman H et al., 1991, Cytometry 12:147-54; Aubele M & WernerM, 1999, Analyt. Cell. Path. 19:53; Shen L et al., 2000, Cancer Res.60:3884).

An alternative model for the observed solid tumor cell heterogeneityderives from the impact of stem cells on tumor development. According tothis model cancer arises from dysregulation of the mechanisms thatcontrol normal tissue development and maintenance. (Beachy et al., 2004,Nature 432:324). During normal animal development, cells of most or alltissues are derived from normal precursors, called stem cells (Morrisonet al., 1997, Cell 88:287-98; Morrison et al., 1997, Curr. Opin.Immunol. 9:216-21; Morrison et al., 1995, Annu. Rev. Cell. Dev. Biol.11:35-71). Stem cells are cells that: (1) have extensive proliferativecapacity; 2) are capable of asymmetric cell division to generate one ormore kinds of progeny with reduced proliferative and/or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. The best-studied example of adult cellrenewal by the differentiation of stem cells is the hematopoietic systemwhere developmentally immature precursors (hematopoietic stem andprogenitor cells) respond to molecular signals to form the varied bloodand lymphoid cell types. Other cells, including cells of the gut, breastductal system, and skin are constantly replenished from a smallpopulation of stem cells in each tissue, and recent studies suggest thatmost other adult tissues also harbor stem cells, including the brain.Tumors derived from a “solid tumor stem cell” (or “cancer stem cell”from a solid tumor) subsequently undergoes chaotic development throughboth symmetric and asymmetric rounds of cell divisions. In this stemcell model, solid tumors contain a distinct and limited (possibly evenrare) subset of cells that share the properties of normal “stem cells”,in that they extensively proliferate and efficiently give rise both toadditional solid tumor stem cells (self-renewal) and to the majority oftumor cells of a solid tumor that lack tumorigenic potential. Indeed,mutations within a long-lived stem cell population may initiate theformation of cancer stem cells that underlie the growth and maintenanceof tumors and whose presence contributes to the failure of currenttherapeutic approaches.

The stem cell nature of cancer was first revealed in the blood cancer,acute myeloid leukemia (AML) (Lapidot et al., 1994, Nature 17:645-8).More recently it has been demonstrated that malignant human breasttumors similarly harbor a small, distinct population of cancer stemcells enriched for the ability to form tumors in immunodeficient mice.An ESA+, CD44+, CD24−/low, Lin− cell population was found to be 50-foldenriched for tumorigenic cells compared to unfractionated tumor cells(Al-Hajj et al., 2003, Proc. Nat'l. Acad. Sci. 100:3983-8). The abilityto prospectively isolate the tumorigenic cancer cells has permittedinvestigation of critical biological pathways that underlietumorigenicity in these cells, and thus promises the development ofbetter diagnostic assays and therapeutics for cancer patients. It istoward this purpose that this invention is directed.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated antibody that specificallybinds to a discoidin/coagulation domain of human DDR2 and has atherapeutic effect on a solid tumor. In certain embodiments the antibodyis a monoclonal antibody. In certain embodiments the antibody is achimeric antibody. In certain embodiments the antibody is a humanizedantibody. In certain embodiments the antibody is a human antibody. Incertain embodiments, the solid tumor is selected from the groupconsisting of a breast tumor, colorectal tumor, lung tumor, pancreatictumor, prostate tumor, and a head and neck tumor.

The present invention provides a pharmaceutical composition comprisingan isolated antibody that specifically binds to a discoidin/coagulationdomain of human DDR2 and a pharmaceutically acceptable vehicle. Incertain embodiments the antibody is a monoclonal antibody. In certainembodiments the antibody is a chimeric antibody. In certain embodimentsthe antibody is a humanized antibody. In certain embodiments he antibodyis a human antibody.

The present invention further provides a method of treating cancer, themethod comprising: administering to a patient with a solid tumor ofepithelial origin a therapeutically effective amount of an antibody thatspecifically binds to a discoidin/coagulation domain of human DDR2. Incertain embodiments the antibody is a monoclonal antibody. In certainembodiments the antibody is a chimeric antibody. In certain embodimentsthe antibody is a humanized antibody. In certain embodiments theantibody is a human antibody. In certain embodiments, the antibody isconjugated to a cytotoxic moiety. In certain embodiments, the method oftreating cancer further comprises administering at least one additionaltherapeutic agent appropriate for effecting combination therapy. Incertain embodiments, the solid tumor is selected from the groupconsisting of a breast tumor, colorectal tumor, lung tumor, pancreatictumor, prostate tumor, and a head and neck tumor.

The present invention provides an isolated antibody that specificallybinds to human DDR2 within a region comprising amino acids 24 to 241 andhas a therapeutic effect on a solid tumor. In certain embodiments theantibody is a monoclonal antibody. In certain embodiments the antibodyis a chimeric antibody. In certain embodiments the antibody is ahumanized antibody. In certain embodiments the antibody is a humanantibody. In certain embodiments, the solid tumor is selected from thegroup consisting of a breast tumor, colorectal tumor, lung tumor,pancreatic tumor, prostate tumor, and a head and neck tumor.

The present invention provides a pharmaceutical composition comprisingan isolated antibody that specifically binds to human DDR2 within aregion comprising amino acids 24 to 241 and a pharmaceuticallyacceptable vehicle. In certain embodiments the antibody is a monoclonalantibody. In certain embodiments the antibody is a chimeric antibody. Incertain embodiments the antibody is a humanized antibody. In certainembodiments the antibody is a human antibody.

The present invention further provides a method of treating cancer, themethod comprising: administering to a patient having a solid tumor ofepithelial origin a therapeutically effective amount of an antibody thatspecifically binds to human DDR2 within a region comprising amino acids24 to 241. In certain embodiments the antibody is a monoclonal antibody.In certain embodiments the antibody is a chimeric antibody. In certainembodiments the antibody is a humanized antibody. In certain embodimentsthe antibody is a human antibody. In certain embodiments, the antibodyis conjugated to a cytotoxic moiety. In certain embodiments, the methodof treating cancer further comprises administering at least oneadditional therapeutic agent appropriate for effecting combinationtherapy. In certain embodiments, the solid tumor is selected from thegroup consisting of a breast tumor, colorectal tumor, lung tumor,pancreatic tumor, prostate tumor, and a head and neck tumor.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and, together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: DDR2 Antibody 9M58 Recognizes an Epitope within theDiscoidin/coagulation Domain. ELISA using deletion constructs of humanDDR2 (DDR2 24-241; DDR2 24-369; and DDR2 24-399, from left to right)demonstrates that anti-DDR2 antibody 9M58 (left bar) recognizes anepitope between amino acids 24 and 241 of DDR2 within theDiscoidin/coagulation domain of human but not mouse DDR2. In contrast, acontrol antibody that binds to a notch ligand, 21M18 (middle bar), doesnot significantly bind to either human or mouse DDR2 (mDDR2). Anti-HuFc(right bar) detects the presence of DDR2 protein.

FIG. 2: DDR2 Antibodies Reduce Breast Tumor Growth. NOD/SCID mice wereinjected with dissociated PE13 breast tumor cells and treated with oneof two anti-DDR2 antibodies (n=5 each antibody) or PBS (n=5). Treatmentwith 9M57 antibodies (light gray diamonds) had no affect on tumorgrowth; treatment with 9M58 antibodies (dark gray triangles) reducedtumor growth starting on day 35 and by over 50% by day 58 as compared toPBS injected controls (black squares).

FIG. 3: DDR2 Antibodies Reduce Colon Tumor Growth. NOD/SCID mice wereinjected with dissociated C9 colon tumor cells and treated withanti-DDR2 antibodies (n=5) or PBS (n=5). Treatment with 9M58 antibodies(gray diamonds) reduced tumor growth starting on day 33 and by almost50% by day 41 as compared to PBS injected controls (black squares).

FIG. 4: Cancer Stem Cells Express Differential Levels of DDR2. A) PE13breast cancer tumorigenic cells (TG: black left bar) express elevatedrelative levels of DDR2 compared to non-tumorigenic tumor cells (NTG:gray right bar). B) C4 colon cancer tumorigenic cells (C4-TG: black bar)express lower relative levels of DDR2 compared to non-tumorigenic tumorcells (C4-NTG: gray bar). In contrast, C6 colon cancer tumorigenic cells(C6-TG: black bar) express elevated relative levels of DDR2 compared tonon-tumorigenic tumor cells (C6-NTG: gray bar).

FIG. 5: DDR2 Antibodies Delay Breast Tumor Recurrence After CombinationTreatment With Taxol. Tumor volume (mm³) over time (days) in NOD/SCIDmice injected with PE13 breast tumor cells and treated with controlantibodies (squares) or 9M58 anti-DDR2 antibodies (triangles) aftercombination therapy with antibodies and taxol. Animals treated with 9M58antibodies show delayed reoccurrence of tumors following combinationtaxol administration compared to animals treated with controlantibodies.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. Incertain embodiments, antibodies of the present invention includeantagonist antibodies that specifically bind to a cancer stem cellmarker protein and interfere with, for example, ligand binding, receptordimerization, expression of a cancer stem cell marker protein, and/ordownstream signaling of a cancer stem cell marker protein. In certainembodiments, disclosed antibodies include agonist antibodies thatspecifically bind to a cancer stem cell marker protein and promote, forexample, ligand binding, receptor dimerization, and/or signaling by acancer stem cell marker protein. In certain embodiments, disclosedantibodies do not interfere with or promote the biological activity of acancer stem cell marker protein but inhibit tumor growth by, forexample, antibody internalization and/or recognized by the immunesystem. As used herein, the term “antibody” encompasses intactpolyclonal antibodies, intact monoclonal antibodies, antibody fragments(such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv)mutants, multispecific antibodies such as bispecific antibodiesgenerated from at least two intact antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen determination portion of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibodies exhibit the desired biological activity. An antibodycan be of any the five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2), based on the identity of their heavy-chainconstant domains referred to as alpha, delta, epsilon, gamma, and mu,respectively. The different classes of immunoglobulins have differentand well known subunit structures and three-dimensional configurations.Antibodies can be naked or conjugated to other molecules such as toxins,radioisotopes, etc.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

An “Fv antibody” refers to the minimal antibody fragment that contains acomplete antigen-recognition and -binding site either as two-chains, inwhich one heavy and one light chain variable domain form a non-covalentdimer, or as a single-chain (scFv), in which one heavy and one lightchain variable domain are covalently linked by a flexible peptide linkerso that the two chains associate in a similar dimeric structure. In thisconfiguration the complementary determining regions (CDRs) of eachvariable domain interact to define the antigen-binding specificity ofthe Fv dimer. Alternatively a single variable domain (or half of an Fv)can be used to recognize and bind antigen, although generally with loweraffinity.

A “monoclonal antibody” as used herein refers to homogenous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), singlechain (scFv) mutants, fusion proteins comprising an antibody portion,and any other modified immunoglobulin molecule comprising an antigenrecognition site. Furthermore, “monoclonal antibody” refers to suchantibodies made in any number of manners including but not limited to byhybridoma, phage selection, recombinant expression, and transgenicanimals.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g. murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability. In some instances, the Fvframework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability. Thehumanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any technique known in the art.This definition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

That an antibody “selectively binds” or “specifically binds” means thatthe antibody reacts or associates more frequently, more rapidly, withgreater duration, with greater affinity, or with some combination of theabove to an epitope than with alternative substances, includingunrelated proteins. “Selectively binds” or “specifically binds” means,for instance, that an antibody binds to a protein with a K_(D) of atleast about 0.1 mM, but more usually at least about 1 μM. “Selectivelybinds” or “specifically binds” means at times that an antibody binds toa protein at times with a K_(D) of at least about 0.1 μM or better, andat other times at least about 0.01 μM or better. Because of the sequenceidentity between homologous proteins in different species, specificbinding can include an antibody that recognizes a cancer stem cellmarker protein in more than one species.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

The terms “isolated” or “purified” refer to material that issubstantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A protein(e.g. an antibody) or nucleic acid of the present disclosure that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from open readingframes that naturally flank the gene and encode proteins other thanprotein encoded by the gene. An isolated antibody is separated fromother non-immunoglobulin proteins and from other immunoglobulin proteinswith different antigen binding specificity. It can also mean that thenucleic acid or protein is in some embodiments at least 80% pure, insome embodiments at least 85% pure, in some embodiments at least 90%pure, in some embodiments at least 95% pure, and in some embodiments atleast 99% pure.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancers.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell”, “tumor stem cell”, or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells”, “tumor stem cells” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.Solid tumor stem cells differ from the “cancer stem line” provided byU.S. Pat. No. 6,004,528. In that patent, the “cancer stem line” isdefined as a slow growing progenitor cell type that itself has fewmutations but which undergoes symmetric rather than asymmetric celldivisions as a result of tumorigenic changes that occur in the cell'senvironment. This “cancer stem line” hypothesis thus proposes thathighly mutated, rapidly proliferating tumor cells arise largely as aresult of an abnormal environment, which causes relatively normal stemcells to accumulate and then undergo mutations that cause them to becometumor cells. U.S. Pat. No. 6,004,528 proposes that such a model can beused to enhance the diagnosis of cancer. The solid tumor stem cell modelis fundamentally different from the “cancer stem line” model and as aresult exhibits utilities not offered by the “cancer stem line” model.First, solid tumor stem cells are not “mutationally spared.” The“mutationally spared cancer stem line” described by U.S. Pat. No.6,004,528 can be considered a pre-cancerous lesion, while solid tumorstem cells are cancer cells that may themselves contain the mutationsthat are responsible for tumorigenesis starting at the pre-cancerousstage through later stage cancer. That is, solid tumor stem cells(“cancer stem cells”) would be included among the highly mutated cellsthat are distinguished from the “cancer stem line” in U.S. Pat. No.6,004,528. Second, the genetic mutations that lead to cancer can belargely intrinsic within the solid tumor stem cells as well as beingenvironmental. The solid tumor stem cell model predicts that isolatedsolid tumor stem cells can give rise to additional tumors upontransplantation (thus explaining metastasis) while the “cancer stemline” model would predict that transplanted “cancer stem line” cellswould not be able to give rise to a new tumor, since it was theirabnormal environment that was tumorigenic. Indeed, the ability totransplant dissociated, and phenotypically isolated human solid tumorstem cells to mice (into an environment that is very different from thenormal tumor environment) where they still form new tumors distinguishesthe present invention from the “cancer stem line” model. Third, solidtumor stem cells likely divide both symmetrically and asymmetrically,such that symmetric cell division is not an obligate property. Fourth,solid tumor stem cells can divide rapidly or slowly, depending on manyvariables, such that a slow proliferation rate is not a definingcharacteristic.

The terms “cancer cell”, “tumor cell” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells).

As used herein “tumorigenic” refers to the functional features of asolid tumor stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor. These properties of self-renewal and proliferation to generateall other tumor cells confer on the cancer stem cells of this inventionthe ability to form palpable tumors upon serial transplantation into animmunocompromised mouse compared to the majority of tumor cells that areunable to form tumors upon the serial transplantation. Tumor cells, i.e.non-tumorigenic tumor cells, may form a tumor upon transplantation intoan immunocompromised mouse a limited number of times (for example one ortwo times) after obtaining the tumor cells from a solid tumor.

As used herein, the terms “stem cell cancer marker(s)”, “cancer stemcell marker(s)”, “tumor stem cell marker(s)”, or “solid tumor stem cellmarker(s)” refer to a gene or genes or a protein, polypeptide, orpeptide expressed by the gene or genes whose expression level, alone orin combination with other genes, is correlated with the presence oftumorigenic cancer cells compared to non-tumorigenic cells. Thecorrelation can relate to either an increased or decreased expression ofthe gene (e.g. increased or decreased levels of mRNA or the peptideencoded by the gene).

As used herein, the terms “biopsy” and “biopsy tissue” refer to a sampleof tissue or fluid that is removed from a subject for the purpose ofdetermining if the sample contains cancerous tissue. In someembodiments, biopsy tissue or fluid is obtained because a subject issuspected of having cancer, and the biopsy tissue or fluid is thenexamined for the presence or absence of cancer.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that can be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

“Prodrug” refers to a derivative of a therapeutically effective compoundthat requires a transformation within the body to produce thetherapeutically effective compound. Prodrugs can be pharmacologicallyinactive until converted to the therapeutically effective parentcompound.

The term “therapeutically effective amount” or “therapeutic effect”refers to an amount of an antibody, polypeptide, polynucleotide, smallorganic molecule, or other drug effective to “treat” a disease ordisorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the drug has a therapeutic effectand as such can reduce the number of cancer cells; decrease tumorigenicfrequency or decrease tumorigenic capacity; reduce the tumor size;inhibit or stop cancer cell infiltration into peripheral organsincluding, for example, the spread of cancer into soft tissue and bone;inhibit and stop tumor metastasis; inhibit and stop tumor growth;relieve to some extent one or more of the symptoms associated with thecancer, reduce morbidity and mortality; improve quality of life; or acombination of such effects. Methods to determine tumorigenic frequencyor capacity are demonstrated in copending application U.S. Ser. No.11/776,935. To the extent the drug prevents growth and/or kills existingcancer cells, it can be referred to as cytostatic and/or cytotoxic.

As used herein, “providing a diagnosis” or “diagnostic information”refers to any information that is useful in determining whether apatient has a disease or condition and/or in classifying the disease orcondition into a phenotypic category or any category having significancewith regards to the prognosis of or likely response to treatment (eithertreatment in general or any particular treatment) of the disease orcondition. Similarly, diagnosis refers to providing any type ofdiagnostic information, including, but not limited to, whether a subjectis likely to have a condition (such as a tumor), information related tothe nature or classification of a tumor as for example a high risk tumoror a low risk tumor, information related to prognosis and/or informationuseful in selecting an appropriate treatment. Selection of treatment caninclude the choice of a particular chemotherapeutic agent or othertreatment modality such as surgery or radiation or a choice aboutwhether to withhold or deliver therapy.

As used herein, the terms “providing a prognosis,” “prognosticinformation,” or “predictive information” refer to providing informationregarding the impact of the presence of cancer (e.g., as determined bythe diagnostic methods of the present invention) on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood of gettingcancer, and the risk of metastasis).

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. A subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including, for example, the spread of cancer into softtissue and bone; inhibition of or an absence of tumor metastasis;inhibition or an absence of tumor growth; relief of one or more symptomsassociated with the specific cancer; reduced morbidity and mortality;improvement in quality of life; or some combination of effects.

As used herein, the terms “polynucleotide” or “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA. The term “gene” refers to a nucleic acid (e.g., DNA) sequencethat comprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns cancontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide. In addition to containing introns, genomic forms ofa gene can also include sequences located on both the 5′ and 3′ end ofthe sequences that are present on the RNA transcript. These sequencesare referred to as “flanking” sequences or regions (these flankingsequences are located 5′ or 3′ to the non-translated sequences presenton the mRNA transcript). The 5′ flanking region can contain regulatorysequences such as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region can contain sequencesthat direct the termination of transcription, post transcriptionalcleavage and polyadenylation.

The term “recombinant” when used with reference to a cell, nucleic acid,protein or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. Thus, e.g.,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and introduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. Similarly, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as depicted above.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments. Unless otherwise provided,ligation can be accomplished using known buffers and conditions with 10units to T4 DNA ligase (“ligase”) per 0.5 ug of approximately equimolaramounts of the DNA fragments to be ligated. Ligation of nucleic acid canserve to link two proteins together in-frame to produce a singleprotein, or fusion protein.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “polypeptide,” “peptide,” “protein,” and “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. One ofskill will recognize that in certain contexts each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, silentvariations of a nucleic acid which encodes a polypeptide is implicit ina described sequence with respect to the expression product, but notwith respect to actual probe sequences. As to amino acid sequences, oneof skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” including where the alteration results in the substitution ofan amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. Such conservatively modified variants are in additionto and do not exclude polymorphic variants, interspecies homologs, andalleles of the invention. Typically conservative substitutionsinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

The term “epitope tagged” as used herein refers to a chimericpolypeptide comprising a cancer stem cell marker protein, or a domainsequence or portion thereof, fused to an “epitope tag”. The epitope tagpolypeptide comprises enough amino acid residues to provide an epitopefor recognition by an antibody, yet is short enough such that it doesnot interfere with the activity of the cancer stem cell marker protein.Suitable epitope tags generally have at least six amino acid residues,usually between about 8 to about 50 amino acid residues, and at timesbetween about 10 to about 20 residues. Commonly used epitope tagsinclude Fc, HA, His, and FLAG tags.

The present invention provides an isolated antibody that specificallybinds to a discoidin/coagulation domain of human DDR2 and has atherapeutic effect on a solid tumor. In certain embodiments the antibodyis a monoclonal antibody. In certain embodiments the antibody is achimeric antibody. In certain embodiments the antibody is a humanizedantibody. In certain embodiments the antibody is a human antibody. Incertain embodiments, the solid tumor is selected from the groupconsisting of a breast tumor, colorectal tumor, lung tumor, pancreatictumor, prostate tumor, and a head and neck tumor.

The present invention provides a pharmaceutical composition comprisingan isolated antibody that specifically binds to a discoidin/coagulationdomain of human DDR2 and a pharmaceutically acceptable vehicle. Incertain embodiments the antibody is a monoclonal antibody. In certainembodiments the antibody is a chimeric antibody. In certain embodimentsthe antibody is a humanized antibody. In certain embodiments he antibodyis a human antibody.

The present invention further provides a method of treating cancer, themethod comprising: (a) identifying a patient having a solid tumor ofepithelial origin; and (b) administering to the patient atherapeutically effective amount of an antibody that specifically bindsto a discoidin/coagulation domain of human DDR2. In certain embodimentsthe antibody is a monoclonal antibody. In certain embodiments theantibody is a chimeric antibody. In certain embodiments the antibody isa humanized antibody. In certain embodiments the antibody is a humanantibody. In certain embodiments, the antibody is conjugated to acytotoxic moiety. In certain embodiments, the method of treating cancerfurther comprises administering at least one additional therapeuticagent appropriate for effecting combination therapy. In certainembodiments, the solid tumor is selected from the group consisting of abreast tumor, colorectal tumor, lung tumor, pancreatic tumor, prostatetumor, and a head and neck tumor.

The present invention provides an isolated antibody that specificallybinds to human DDR2 within a region comprising amino acids 24 to 241 andhas a therapeutic effect on a solid tumor. In certain embodiments theantibody is a monoclonal antibody. In certain embodiments the antibodyis a chimeric antibody. In certain embodiments the antibody is ahumanized antibody. In certain embodiments the antibody is a humanantibody. In certain embodiments, the solid tumor is selected from thegroup consisting of a breast tumor, colorectal tumor, lung tumor,pancreatic tumor, prostate tumor, and a head and neck tumor.

The present invention provides a pharmaceutical composition comprisingan isolated antibody that specifically binds to human DDR2 within aregion comprising amino acids 24 to 241 and a pharmaceuticallyacceptable vehicle. In certain embodiments the antibody is a monoclonalantibody. In certain embodiments the antibody is a chimeric antibody. Incertain embodiments the antibody is a humanized antibody. In certainembodiments the antibody is a human antibody.

The present invention further provides a method of treating cancer, themethod comprising: (a) identifying a patient having a solid tumor ofepithelial origin; and (b) administering to the patient atherapeutically effective amount of an antibody that specifically bindsto human DDR2 within a region comprising amino acids 24 to 241. Incertain embodiments the antibody is a monoclonal antibody. In certainembodiments the antibody is a chimeric antibody. In certain embodimentsthe antibody is a humanized antibody. In certain embodiments theantibody is a human antibody. In certain embodiments, the antibody isconjugated to a cytotoxic moiety. In certain embodiments, the method oftreating cancer further comprises administering at least one additionaltherapeutic agent appropriate for effecting combination therapy. Incertain embodiments, the solid tumor is selected from the groupconsisting of a breast tumor, colorectal tumor, lung tumor, pancreatictumor, prostate tumor, and a head and neck tumor.

Like the tissues in which they originate, solid tumors consist of aheterogeneous population of cells. That the majority of these cells lacktumorigenicity suggested that the development and maintenance of solidtumors also relies on a small population of stem cells (i.e.,tumorigenic cancer cells) with the capacity to proliferate andefficiently give rise both to additional tumor stem cells (self-renewal)and to the majority of more differentiated tumor cells that lacktumorigenic potential (i.e., non-tumorigenic cancer cells). The conceptof cancer stem cells was first introduced soon after the discovery ofHSC and was established experimentally in acute myelogenous leukemia(AML) (Park et al., 1971, J. Natl. Cancer Inst. 46:411-22; Lapidot etal., 1994, Nature 367:645-8; Bonnet & Dick, 1997, Nat. Med. 3:730-7;Hope et al., 2004, Nat. Immunol. 5:738-43). Stem cells from solid tumorshave more recently been isolated based on their expression of a uniquepattern of cell-surface receptors and on the assessment of theirproperties of self-renewal and proliferation in culture and in xenograftanimal models. An ESA+ CD44+ CD24−/low Lineage-population greater than50-fold enriched for the ability to form tumors relative tounfractionated tumor cells was discovered (Al-Hajj et al., 2003, Proc.Nat'l. Acad. Sci. 100:3983-8). The ability to isolate tumorigenic cancerstem cells from the bulk of non-tumorigenic tumor cells has led to theidentification of cancer stem cell markers, genes with differentialexpression in cancer stem cells compared to non-tumorigenic tumor cellsor normal breast epithelium, using microarray analysis. The presentinvention employs the knowledge of these identified cancer stem cellmarkers to diagnosis and treat cancer.

The cancer stem cell marker of the present invention relates to humandiscoidin domain receptor 2 (DDR2). The discoidin domain receptors DDR1and DDR2 form a subfamily of receptor tyrosine kinases based on thepresence of an extracellular discoidin domain, a domain first identifiedin the slime mold Dictyostelium discoideum that functions in cellaggregation. Collagen serves as the physiological ligand for DDR2, andthis interaction both inhibits fibrillogenesis of collagen and regulatesexpression of matrix-metalloproteases (MMP), enzymes that cleave nativefibrillar collagen (Vogel, 1999, FASEB 13:S77; Xu et al., 2005, J. Biol.Chem. 280:548-55; Mihai et al., 2006, J. Mol. Biol. 361:864-76). Therole of DDR2 in regulation of the extracellular matrix suggests thatdysregulation of DDR signaling may contribute to human carcinogenesis,including invasion and metastasis.

DRR2 signaling regulates proliferation of various cell populationsincluding chondrocytes and fibroblasts (Labrador et al., 2001, EMBO Rep.2:446-52). DDR2 is induced in hepatic stellate cells in response tocollagen during liver injury, and overexpression of DDR enhanced hepaticstellate cell proliferation, activated expression of MMP-2, and enhancedcellular invasion through Matrigel (Olaso et al., 2001, J. Clin. Invest.108:1369-78). DDR activation and adhesion in response to collagen mayrequire Wnt and G-protein signaling (Dejmek et al., 2003, Int. J. Cancer103:344-51).

DDR receptors are implicated in cancer. DDR1 is overexpressed innumerous human tumors including breast, ovarian, esophageal, and braincancers (Barker et al., 1995, Oncogene 11:569-75; Laval et al., 1994,Cell Growth Diff: 5:1173-83; Nemoto et al., 1997, Pathobiol. 65:165-203;Weiner et al., 1996, Pediatr. Neurosurg. 25:64-72; Weiner et al., 2000,Neurosurgery 47:1400-9; Heinzelmann et al., 2004, 10:4427-36). DDR1 andDDR2 have mutually exclusive expression in ovarian and lung tumors, withtranscripts for DDR1 in highly invasive tumor cells and transcripts forDDR2 detected in the surrounding stromal cells (Alves et al., 1995,Oncogene 10:609-18). Furthermore, DDR2 expression is associated withinvasive mammary carcinomas (Evitmova et al., 2003, Tumour Biol.24:189-98). Thus the identification of DDR2 as a marker of cancer stemcells suggests that targeting these receptors may prove therapeuticallyeffective in treating human cancers.

Thus the present invention provides the cancer stem cell marker DDR2,the expression of which can be analyzed to diagnose or monitor cancer.In some embodiments, expression of a cancer stem cell marker isdetermined by polynucleotide expression such as, for example, mRNAencoding the cancer stem cell marker. The polynucleotide can be detectedand quantified by any of a number of means well known to those of skillin the art. In some embodiments, mRNA encoding a cancer stem cell markeris detected by in situ hybridization of tissue sections from, forexample, a patient biopsy. In some embodiments, RNA is isolated from atissue and detected by, for example, Northern blot, quantitative RT-PCR,or microarrays. For example, total RNA can be extracted from a tissuesample and primers that specifically hybridize and amplify a cancer stemcell marker can be used to detect expression of a cancer stem cellmarker polynucleotide using RT-PCR.

In certain embodiments, expression of a cancer stem cell marker can bedetermined by detection of the corresponding polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. In some embodiments, a cancerstem cell marker polypeptide is detected using analytic biochemicalmethods such as, for example, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC) or thinlayer chromatography (TLC). The isolated polypeptide can also besequenced according to standard techniques. In some embodiments, acancer stem cell marker protein is detected with antibodies raisedagainst the protein using, for example, immunofluorescence orimmunohistochemistry on tissue sections. Alternatively antibodiesagainst a cancer stem cell marker can detect expression using, forexample, ELISA, FACS, Western blot, immunoprecipitation or proteinmicroarrays. For example, cancer stem cells can be isolated from apatient biopsy and expression of a cancer stem cell marker proteindetected with fluorescently labeled antibodies using FACS. In anothermethod, the cells expressing a cancer stem cell marker can be detectedin vivo using labeled antibodies in typical imaging system. For example,antibodies labeled with paramagnetic isotopes can be used for magneticresonance imaging (MRI).

In some embodiments of the present invention, a diagnostic assaycomprises determining the expression or not of a cancer stem cell markerin tumor cells using, for example, immunohistochemistry, in situhybridization, or RT-PCR. In other embodiments, a diagnostic assaycomprises determining expression levels of a cancer stem cell markerusing, for example, quantitative RT-PCR. In some embodiments, adiagnostic assay further comprises determining expression levels of acancer stem cell marker compared to a control tissue such as, forexample, normal epithelium.

Detection of a cancer stem cell marker expression can then be used toprovide a prognosis and select a therapy. A prognosis can be based onany known risk expression of a cancer stem cell marker indicates.Furthermore, detection of a cancer stem cell marker can be used toselect an appropriate therapy including, for example, treatment withantibodies against the detected cancer stem cell marker protein. Incertain embodiments, the antibody specifically binds to theextracellular domain of the cancer stem cell marker protein human DDR2.

In the context of the present invention, a suitable antibody is an agentthat can have one or more of the following effects, for example:interfere with the expression of a cancer stem cell marker; interferewith activation of a cancer stem cell signal transduction pathway by,for example, sterically inhibiting interactions between a cancer stemcell marker and its ligand, receptor or co-receptors; activate a cancerstem cell signal transduction pathway by, for example, acting as aligand or promoting the binding of an endogenous ligand; or bind to acancer stem cell marker and inhibit tumor cell proliferation.

In certain embodiments, antibodies against a cancer stem cell marker actextracellularly to modulate the function of a cancer stem cell markerprotein. In some embodiments, extracellular binding of an antibodyagainst a cancer stem cell marker can inhibit the signaling of a cancerstem cell marker protein by, for example, inhibiting intrinsicactivation (e g kinase activity) of a cancer stem cell marker and/or bysterically inhibiting the interaction, for example, of a cancer stemcell marker with its ligand, with its receptor, with a co-receptor, orwith the extracellular matrix. In some embodiments, extracellularbinding of an antibody against a cancer stem cell marker candownregulate cell-surface expression of a cancer stem cell marker suchas, for example, by internalization of a cancer stem cell marker proteinor decreasing cell surface trafficking of a cancer stem cell marker. Insome embodiments, extracellular binding of an antibody against a cancerstem cell marker can promote the signaling of a cancer stem cell markerprotein by, for example, acting as a decoy ligand or increasing ligandbinding.

In certain embodiments, antibodies against a cancer stem cell markerbind to a cancer stem cell marker protein and have one or more of thefollowing effects: inhibit proliferation of tumor cells, trigger celldeath of tumor cells, or prevent metastasis of tumor cells. In certainembodiments, antibodies against a cancer stem cell marker trigger celldeath via a conjugated toxin, chemotherapeutic agent, radioisotope, orother such agent. For example, an antibody against a cancer stem cellmarker is conjugated to a toxin that is activated in tumor cellsexpressing the cancer stem cell marker by protein internalization. Incertain embodiments, antibodies against a cancer stem cell markermediate cell death of a cell expressing the cancer stem cell markerprotein via antibody-dependent cellular cytotoxicity (ADCC). ADCCinvolves cell lysis by effector cells that recognize the Fc portion ofan antibody. Many lymphocytes, monocytes, tissue macrophages,granulocytes and eosinophiles, for example, have Fc receptors and canmediate cytolysis (Dillman, 1994, J. Clin. Oncol. 12:1497).

In certain embodiments, antibodies against a cancer stem cell markertrigger cell death of a cell expressing a cancer stem cell markerprotein by activating complement-dependent cytotoxicity (CDC). CDCinvolves binding of serum complement to the Fc portion of an antibodyand subsequent activation of the complement protein cascade, resultingin cell membrane damage and eventual cell death. Biological activity ofantibodies is known to be determined, to a large extent, by the constantdomains or Fc region of the antibody molecule (Uananue and Benacerraf,Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).Antibodies of different classes and subclasses differ in this respect,as do antibodies of the same subclass but from different species. Ofhuman antibodies, IgM is the most efficient class of antibodies to bindcomplement, followed by IgG1, IgG3, and IgG2 whereas IgG4 appears quitedeficient in activating the complement cascade (Dillman, 1994, J. Clin.Oncol. 12:1497; Jefferis et al., 1998, Immunol. Rev. 163:59-76).According to the present invention, antibodies of those classes havingthe desired biological activity are prepared.

The ability of any particular antibody against a cancer stem cell tomediate lysis of the target cell by complement activation and/or ADCCcan be assayed. The cells of interest are grown and labeled in vitro;the antibody is added to the cell culture in combination with eitherserum complement or immune cells which can be activated by the antigenantibody complexes. Cytolysis of the target cells is detected, forexample, by the release of label from the lysed cells. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibody that is capable ofactivating complement or mediating ADCC in the in vitro test can then beused therapeutically in that particular patient.

In certain embodiments, antibodies against a cancer stem cell marker cantrigger cell death inhibiting angiogenesis. Angiogenesis is the processby which new blood vessels form from pre-existing vessels and is afundamental process required for normal growth, for example, duringembryonic development, wound healing, and in response to ovulation.Solid tumor growth larger than 1-2 mm² also requires angiogenesis tosupply nutrients and oxygen without which tumor cells die. In certainembodiments, an antibody against a cancer stem cell marker targetsvascular cells that express the cancer stem cell marker including, forexample, endothelial cells, smooth muscle cells, or components of theextracellular matrix required for vascular assembly. In certainembodiments, an antibody against a cancer stem cell marker inhibitsgrowth factor signaling required by vascular cell recruitment, assembly,maintenance, or survival.

The antibodies against a cancer stem cell marker find use in thediagnostic and therapeutic methods described herein. In certainembodiments, the antibodies of the present invention are used to detectthe expression of a cancer stem cell marker protein in biologicalsamples such as, for example, a patient tissue biopsy, pleural effusion,or blood sample. Tissue biopsies can be sectioned and protein detectedusing, for example, immunofluorescence or immunohistochemistry. Inaddition, individual cells from a sample can be isolated, and proteinexpression detected on fixed or live cells by FACS analysis. In certainembodiments, antibodies can be used on protein arrays to detectexpression of a cancer stem cell marker, for example, on tumor cells, incell lysates, or in other protein samples. In certain embodiments, theantibodies of the present invention are used to inhibit the growth oftumor cells by contacting the antibodies with tumor cells in in vitrocell based assays, in vivo animal models, etc. In certain embodiments,the antibodies are used to treat cancer in a patient by administering atherapeutically effective amount of an antibody against a cancer stemcell marker.

The antibodies of the present invention can be prepared by anyconventional means known in the art. For example, polyclonal antibodiescan be prepared by immunizing an animal (e.g. a rabbit, rat, mouse,donkey, etc) by multiple subcutaneous or intraperitoneal injections ofthe relevant antigen (a purified peptide fragment, full-lengthrecombinant protein, fusion protein, etc) optionally conjugated tokeyhole limpet hemocyanin (KLH), serum albumin, etc. diluted in sterilesaline and combined with an adjuvant (e.g. Complete or IncompleteFreund's Adjuvant) to form a stable emulsion. The polyclonal antibody isthen recovered from blood, ascites and the like, of an animal soimmunized. Collected blood is clotted, and the serum decanted, clarifiedby centrifugation, and assayed for antibody titer. The polyclonalantibodies can be purified from serum or ascites according to standardmethods in the art including affinity chromatography, ion-exchangechromatography, gel electrophoresis, dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody, can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody, or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments of the present invention, the monoclonal antibodyagainst a cancer stem cell marker is a humanized antibody. Humanizedantibodies are antibodies that contain minimal sequences from non-human(e.g., murine) antibodies within the variable regions. Such antibodiesare used therapeutically to reduce antigenicity and HAMA (humananti-mouse antibody) responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g. mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

In addition, fully human antibodies can be directly prepared usingvarious techniques known in the art. Immortalized human B lymphocytesimmunized in vitro or isolated from an immunized individual that producean antibody directed against a target antigen can be generated (See,e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; andU.S. Pat. No. 5,750,373). Also, the human antibody can be selected froma phage library, where that phage library expresses human antibodies(Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets et al., 1998,Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and Winter, 1991, J.Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Humanantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a cancer stem cell marker. Bispecific antibodies areantibodies that are capable of specifically recognizing and binding atleast two different epitopes. The different epitopes can either bewithin the same molecule (e.g. the same cancer stem cell markerpolypeptide) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a cancer stem cell markeras well as, for example, 1) an effector molecule on a leukocyte such asa T-cell receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16)or 2) a cytotoxic agent as described in detail below. Bispecificantibodies can be intact antibodies or antibody fragments.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tuft et al., J. Immunol. 147:60 (1991))

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to a polypeptide of theinvention (see U.S. Pat. No. 4,946,778). In addition, methods can beadapted for the construction of Fab expression libraries (Huse, et al.,Science 246:1275-1281 (1989)) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor DDR2, or derivatives, fragments, or homologs thereof. Antibodyfragments that contain the idiotypes to a polypeptide of the inventionmay be produced by techniques in the art including, but not limited to:(a) an F(ab′)2 fragment produced by pepsin digestion of an antibodymolecule; (b) an Fab fragment generated by reducing the disulfidebridges of an F(ab′)2 fragment, (c) an Fab fragment generated by thetreatment of the antibody molecule with papain and a reducing agent, and(d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides ofDDR2. In this regard, the variable region may comprise or be derivedfrom any type of mammal that can be induced to mount a humoral responseand generate immunoglobulins against the desired tumor associatedantigen. As such, the variable region of the modified antibodies can be,for example, of human, murine, non-human primate (e.g. cynomolgusmonkeys, macaques, etc.) or lupine origin. In some embodiments both thevariable and constant regions of the modified immunoglobulins are human.In other embodiments the variable regions of compatible antibodies(usually derived from a non-human source) can be engineered orspecifically tailored to improve the binding properties or reduce theimmunogenicity of the molecule. In this respect, variable regions usefulin the present invention can be humanized or otherwise altered throughthe inclusion of imported amino acid sequences.

The variable domains in both the heavy and light chains are altered byat least partial replacement of one or more CDRs and, if necessary, bypartial framework region replacement and sequence changing. Although theCDRs may be derived from an antibody of the same class or even subclassas the antibody from which the framework regions are derived, it isenvisaged that the CDRs will be derived from an antibody of differentclass and preferably from an antibody from a different species. It maynot be necessary to replace all of the CDRs with the complete CDRs fromthe donor variable region to transfer the antigen binding capacity ofone variable domain to another. Rather, it may only be necessary totransfer those residues that are necessary to maintain the activity ofthe antigen binding site. Given the explanations set forth in U.S. Pat.Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within thecompetence of those skilled in the art, either by carrying out routineexperimentation or by trial and error testing to obtain a functionalantibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies, or immunoreactive fragments thereof, in which atleast a fraction of one or more of the constant region domains has beendeleted or otherwise altered so as to provide desired biochemicalcharacteristics such as increased tumor localization or reduced serumhalf-life when compared with an antibody of approximately the sameimmunogenicity comprising a native or unaltered constant region. In someembodiments, the constant region of the modified antibodies willcomprise a human constant region. Modifications to the constant regioncompatible with this invention comprise additions, deletions orsubstitutions of one or more amino acids in one or more domains. Thatis, the modified antibodies disclosed herein may comprise alterations ormodifications to one or more of the three heavy chain constant domains(CH1, CH2 or CH3) and/or to the light chain constant domain (CL). Insome embodiments of the invention modified constant regions wherein oneor more domains are partially or entirely deleted are contemplated. Insome embodiments the modified antibodies will comprise domain deletedconstructs or variants wherein the entire CH2 domain has been removed(ΔCH2 constructs). In some embodiments the omitted constant regiondomain will be replaced by a short amino acid spacer (e.g. 10 residues)that provides some of the molecular flexibility typically imparted bythe absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theCl component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production. Although various Fc receptors and receptorsites have been studied to a certain extent, there is still much whichis unknown about their location, structure and functioning.

While not limiting the scope of the present invention, it is believedthat antibodies comprising constant regions modified as described hereinprovide for altered effector functions that, in turn, affect thebiological profile of the administered antibody. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified antibody thereby increasing tumor localization. In other casesit may be that constant region modifications, consistent with thisinvention, moderate complement binding and thus reduce the serum halflife and nonspecific association of a conjugated cytotoxin. Yet othermodifications of the constant region may be used to eliminate disulfidelinkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. Similarly, modifications to the constant region inaccordance with this invention may easily be made using well knownbiochemical or molecular engineering techniques well within the purviewof the skilled artisan.

It will be noted that the modified antibodies may be engineered to fusethe CH3 domain directly to the hinge region of the respective modifiedantibodies. In other constructs it may be desirable to provide a peptidespacer between the hinge region and the modified CH2 and/or CH3 domains.For example, compatible constructs could be expressed wherein the CH2domain has been deleted and the remaining CH3 domain (modified orunmodified) is joined to the hinge region with a 5-20 amino acid spacer.Such a spacer may be added, for instance, to ensure that the regulatoryelements of the constant domain remain free and accessible or that thehinge region remains flexible. However, it should be noted that aminoacid spacers can, in some cases, prove to be immunogenic and elicit anunwanted immune response against the construct. Accordingly, any spaceradded to the construct be relatively non-immunogenic or, even omittedaltogether if the desired biochemical qualities of the modifiedantibodies may be maintained.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention may be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it can be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. In some embodiments, the antibodies can be conjugatedto radioisotopes, such as ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm,⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷¹Lu, ¹⁸⁶Re and ¹⁸⁸Re using anyone of a number ofwell known chelators or direct labeling. In other embodiments, thedisclosed compositions can comprise antibodies coupled to drugs,prodrugs or lymphokines such as interferon. Conjugates of the antibodyand cytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Conjugatesof an antibody and one or more small molecule toxins, such as acalicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, can also be used.In some embodiments, the modified antibodies can be complexed with otherimmunologically active ligands (e.g. antibodies or fragments thereof)wherein the resulting molecule binds to both the neoplastic cell and aneffector cell such as a T cell.

Conjugate antibodies are composed of two covalently joined antibodies.Such antibodies have, for example, been proposed to target immune cellsto unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins can be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Regardless of how useful quantities are obtained, the antibodies of thepresent invention can be used in any one of a number of conjugated (i.e.an immunoconjugate) or unconjugated forms. Alternatively, the antibodiesof this invention can be used in a nonconjugated or “naked” form toharness the subject's natural defense mechanisms includingcomplement-dependent cytotoxicity (CDC) and antibody dependent cellulartoxicity (ADCC) to eliminate the malignant cells. The selection of whichconjugated or unconjugated modified antibody to use will depend of thetype and stage of cancer, use of adjunct treatment (e.g., chemotherapyor external radiation) and patient condition. It will be appreciatedthat one skilled in the art could readily make such a selection in viewof the teachings herein.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as BIAcore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, and proteinA immunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated byreference herein in its entirety).

In some embodiments, the immunospecificity of an antibody against acancer stem cell marker is determined using ELISA. An ELISA assaycomprises preparing antigen, coating wells of a 96 well microtiter platewith antigen, adding the antibody against a cancer stem cell markerconjugated to a detectable compound such as an enzymatic substrate (e.g.horseradish peroxidase or alkaline phosphatase) to the well, incubatingfor a period of time and detecting the presence of the antigen. In someembodiments, the antibody against a cancer stem cell marker is notconjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the antibody against a cancer stem cell markeris added to the well. In some embodiments, instead of coating the wellwith the antigen, the antibody against a cancer stem cell marker can becoated to the well and a second antibody conjugated to a detectablecompound can be added following the addition of the antigen to thecoated well. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected as wellas other variations of ELISAs known in the art (see e.g. Ausubel et al,eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley &Sons, Inc., New York at 11.2.1).

The binding affinity of an antibody to a cancer stem cell marker antigenand the off-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen (e.g.³H or ¹²⁵I), or fragment or variant thereof, with the antibody ofinterest in the presence of increasing amounts of unlabeled antigenfollowed by the detection of the antibody bound to the labeled antigen.The affinity of the antibody against a cancer stem cell marker and thebinding off-rates can be determined from the data by scatchard plotanalysis. In some embodiments, BIAcore kinetic analysis is used todetermine the binding on and off rates of antibodies against a cancerstem cell marker. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of antibodies from chips with immobilizedcancer stem cell marker antigens on their surface.

In certain embodiments, the invention encompasses isolatedpolynucleotides that encode a polypeptide comprising an antibody, orfragment thereof, against human DDR2. Thus, the term “polynucleotideencoding a polypeptide” encompasses a polynucleotide which includes onlycoding sequences for the polypeptide as well as a polynucleotide whichincludes additional coding and/or non-coding sequences. Thepolynucleotides of the invention can be in the form of RNA or in theform of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and canbe double-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives. The variant of the polynucleotide can be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide. In certain embodiments, thepolynucleotide can have a coding sequence which is a naturally occurringallelic variant of the coding sequence of the disclosed polypeptides. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence that have, for example, a substitution,deletion, or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

In certain embodiments, the present invention provides isolated nucleicacid molecules having a nucleotide sequence at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, andin some embodiments, at least 96%, 97%, 98% or 99% identical to apolynucleotide encoding a polypeptide comprising an antibody, orfragment thereof, against human DDR2.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482 489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against human DDR2. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against human DDR2 protein. Suchmutants include deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors are used to amplify and express DNAencoding cancer stem cell marker polypeptide fusions. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a cancer stem cell markerpolypeptide fusion or a bioequivalent analog operatively linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous and, in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a cancer stem cell marker proteininclude prokaryotes, yeast, insect or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), therelevant disclosure of which is hereby incorporated by reference.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

The present invention provides methods for inhibiting the growth oftumorigenic cells expressing a cancer stem cell marker using theantibodies against a cancer stem cell marker described herein. Incertain embodiments, the method of inhibiting the growth of tumorigeniccells expressing a cancer stem cell marker comprises contacting the cellwith an antibody against a cancer stem cell marker in vitro. Forexample, an immortalized cell line or a cancer cell line that expressesa cancer stem cell marker is cultured in medium to which is added anantibody against the expressed cancer stem cell marker to inhibit cellgrowth. In some embodiments, tumor cells comprising tumor stem cells areisolated from a patient sample such as, for example, a tissue biopsy,pleural effusion, or blood sample and cultured in medium to which isadded an antibody against a cancer stem cell marker to inhibit cellgrowth.

In some embodiments, the method of inhibiting the growth of tumorigeniccells expressing a cancer stem cell marker comprises contacting the cellwith an antibody against a cancer stem cell marker in vivo. In certainembodiments, contacting a tumorigenic cell with an antibody against acancer stem cell marker is undertaken in an animal model. For example,xenografts expressing a cancer stem cell marker are grown inimmunocompromised mice (e.g. NOD/SCID mice) that are administered anantibody against a cancer stem cell marker to inhibit tumor growth. Insome embodiments, cancer stem cells that express a cancer stem cellmarker are isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice that are then administered an antibody againstthe cancer stem cell marker to inhibit growth of a solid tumor. In someembodiments, the antibody against a cancer stem cell marker isadministered at the same time or shortly after introduction oftumorigenic cells into the animal to prevent tumor growth. In someembodiments, the antibody against a cancer stem cell marker isadministered as a therapeutic after the tumorigenic cells have grown toa specified size.

The present invention further provides pharmaceutical compositionscomprising antibodies that target a cancer stem cell marker. Thesepharmaceutical compositions find use in inhibiting growth of a solidtumor and treating cancer in human patients.

Formulations are prepared for storage and use by combining a purifiedantibody of the present invention with a pharmaceutically acceptablevehicle (e.g. carrier, excipient) (Remington, The Science and Practiceof Pharmacy 20th Edition Mack Publishing, 2000). Suitablepharmaceutically acceptable vehicles include, but are not limited to,nontoxic buffers such as phosphate, citrate, and other organic acids;salts such as sodium chloride; antioxidants including ascorbic acid andmethionine; preservatives (e.g. octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens, such asmethyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (e.g. lessthan about 10 amino acid residues); proteins such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosaccharides, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and non-ionic surfactants such as TWEEN orpolyethylene glycol (PEG).

The pharmaceutical composition of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

Pharmaceutical formulations include antibodies of the present inventioncomplexed with liposomes (Epstein, et al., 1985, Proc. Natl. Acad. Sci.USA 82:3688; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030;and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Someliposomes can be generated by the reverse phase evaporation with a lipidcomposition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The antibodies can also be entrapped in microcapsules. Suchmicrocapsules are prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

In some embodiments, the treatment involves the combined administrationof an antibody of the present invention and a chemotherapeutic agent orcocktail of multiple different chemotherapeutic agents. Treatment withan antibody can occur prior to, concurrently with, or subsequent toadministration of chemotherapies. Chemotherapies contemplated by theinvention include chemical substances or drugs which are known in theart and are commercially available, such as Doxorubicin, 5-Fluorouracil,Cytosine arabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan,Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine andCarboplatin. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992).

In other embodiments, the treatment involves the combined administrationof an antibody of the present invention and radiation therapy. Treatmentwith the antibody can occur prior to, concurrently with, or subsequentto administration of radiation therapy. Any dosing schedules for suchradiation therapy can be used as determined by the skilled practitioner.

In other embodiments, the treatment can involve the combinedadministration of antibodies of the present invention with otherantibodies against additional tumor associated antigens including, butnot limited to, antibodies that bind to the EGF receptor (EGFR)(Erbitux® (Bristol-Myers Squibb Company, Princeton, N.J.)), the erbB2receptor (HER2) (Herceptin® (Genentech, Inc., South San Francisco,Calif.)), and vascular endothelial growth factor (VEGF) (Avastin®(Genentech, Inc., South San Francisco, Calif.)). Furthermore, treatmentcan include administration of one or more cytokines, can be accompaniedby surgical removal of cancer cells or any other therapy deemednecessary by a treating physician.

For the treatment of the disease, the appropriate dosage of an antibodyof the present invention depends on the type of disease to be treated,the severity and course of the disease, the responsiveness of thedisease, whether the antibody is administered for therapeutic orpreventative purposes, previous therapy, patient's clinical history, andso on all at the discretion of the treating physician. The antibody canbe administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g. reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antibody. The administeringphysician can easily determine optimum dosages, dosing methodologies andrepetition rates. In general, dosage is from 0.01 μg to 100 mg per kg ofbody weight, and can be given once or more daily, weekly, monthly oryearly. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues.

The present invention provides kits comprising the antibodies describedherein and that can be used to perform the methods described herein. Incertain embodiments, a kit comprises at least one purified antibodyagainst a cancer stem cell marker in one or more containers. In someembodiments, the kits contain all of the components necessary and/orsufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results. One skilled in the art willreadily recognize that the disclosed antibodies of the present inventioncan be readily incorporated into one of the established kit formatswhich are well known in the art.

Embodiments of the present disclosure can be further defined byreference to the following examples, which describe in detailpreparation of antibodies of the present disclosure and methods forusing antibodies of the present disclosure. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of thepresent disclosure. Wherever possible, the same reference numbers willbe used throughout the drawings to refer to the same or like parts. Asused herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an antibody” includes aplurality of such antibodies or one or more antibodies and equivalentsthereof known to those skilled in the art. Furthermore, all numbersexpressing quantities of ingredients, reaction conditions, purity,polypeptide and polynucleotide lengths, and so forth, used in thespecification, are modified by the term “about,” unless otherwiseindicated. Accordingly, the numerical parameters set forth in thespecification and claims are approximations that may vary depending uponthe desired properties of the present invention.

EXAMPLES Example 1 Production of DDR2 Antibodies Antigen Production

Recombinant polypeptide fragments of the extracellular domain of humanDDR2 were generated as antigens for antibody production. Standardrecombinant DNA technology was used to isolate polynucleotides encodingamino acids 1-399 of DDR2 (SEQ ID NO: 1). This polynucleotide wasligated in-frame N-terminal to either a human Fc-tag or histidine-tagand cloned into a transfer plasmid vector for baculovirus mediatedexpression in insect cells. Standard transfection, infection, and cellculture protocols were used to produce recombinant insect cellsexpressing the corresponding DDR2 polypeptide (O'Reilley et al.,Baculovirus expression vectors: A Laboratory Manual, Oxford: OxfordUniversity Press (1994)).

Cleavage of the endogenous signal sequence of human DDR2 wasapproximated using cleavage prediction software SignalP 3.0, however theactual in vivo cleavage point can differ by a couple of amino acidseither direction. The predicated cleavage of DDR2 is between amino acids23 and 24, thus DDR2 antigen protein comprises approximately amino acid24 through amino acid 399. Antigen protein was purified from insect cellconditioned medium using Protein A and Ni⁺⁺-chelate affinitychromatography. Purified antigen protein was dialyzed against PBS(pH=7), concentrated to approximately 1 mg/ml, and sterile filtered inpreparation for immunization.

Immunization

Mice (n=3) were immunized with purified DDR2 antigen protein (AntibodySolutions; Mountain View, Calif.) using standard techniques. Blood fromindividual mice was screened approximately 70 days after initialimmunization for antigen recognition using ELISA and FACS analysis(described in detail below). The two animals with the highest antibodytiters were selected for final antigen boost after which spleen cellswere isolated for hybridoma production. Hybridoma cells were plated at 1cell per well in 96 well plates, and the supernatant from each wellscreened by ELISA and FACS analysis against antigen protein. Severalhybridomas with high antibody titer were selected and scaled up instatic flask culture. Antibodies were purified from the hybridomasupernatant using protein A or protein G agarose chromatography.Purified monoclonal antibodies were again tested by FACS and areisotyped to select for IgG and IgM antibodies.

Epitope Mapping

To identify antibodies that recognize specific regions of the DDR2extracellular domain, epitope mapping was performed. Mammalianexpression plasmid vectors comprising a CMV promoter upstream ofpolynucleotides encoding a series of deletion fragments of theextracellular domain of DDR2 fused to Fc protein were generated usingstandard recombinant DNA technology. These recombinant fusion proteinswere expressed in transiently transfected HEK 293 cells from whichconditioned medium was collected twenty-four to forty-eight hourspost-transfection for ELISA. The Fc fusion proteins in the conditionedmedia were bound to an ELISA plate coated with anti-human Fc (gammachain specific), which was then incubated with 9M58, an anti-DDR2antibody, or 21M18, an anti-Notch ligand antibody, or a controlmonoclonal antibody. After washing, the bound antibodies were probedwith HRP conjugated anti-mouse antibody. To verify equivalent binding ofthe various DDR2 Fc fusion proteins, the ELISA plate was also probedwith HRP conjugated anti-human Fc antibodies. Bound HRP concentrationswere determined at A₄₅₀nm using an ELISA substrate. As shown in FIG. 1,monoclonal antibody 9M58 recognizes an epitope within theDiscoidin/coagulation domain and specifically recognizes human DDR2, notmurine DDR2 Fc fusion protein.

The SPOTs system (Sigma Genosys, The Woodlands, Tex.) can also be usedto identify specific epitopes within the extracellular domainsrecognized by an antibody against DDR2. In this method, a series of10-residue linear peptides overlapping by one amino acid and coveringthe entire DDR2 extracellular domain are synthesized and covalentlybound to a cellulose membrane by the SPOT synthesis technique. Themembrane is preincubated for 8 hours at room temperature with blockingbuffer and hybridized with antibody overnight at 4° C. The membrane isthen washed, incubated with a secondary antibody conjugated tohorseradish peroxidase (HRP) (Amersham Bioscience, Piscataway, N.J.),re-washed, and visualized with signal development solution containing3-amino-9-ethylcarbazole. Specific epitopes recognized by an antibodyare thus determined.

FACS Analysis

To select monoclonal antibodies produced by hybridomas clones thatrecognize native cell-surface DDR2 protein, FACs analysis can be used.HEK293 cells are transfected with an expression vector encoding afull-length cDNA encoding DDR2 with a vector expressing GFP. Twenty-fourto 48-hours post-transfection, cells are collected in suspension andincubated on ice with anti-DDR2 antibodies or control IgG antibodies todetect background antibody binding. The cells are washed and primaryantibodies detected with anti-mouse secondary antibodies conjugated to afluorescent chromophore. Labeled cells are then sorted by FACS toidentify anti-DDR2 antibodies that specifically recognize DDR2 cellsurface protein expression.

Chimeric Antibodies

After monoclonal antibodies that specifically recognize human DDR2 areidentified, these antibodies can be modified to overcome the humananti-mouse antibody (HAMA) immune response when rodent antibodies areused as therapeutics agents. The variable regions of the heavy-chain andlight-chain of the selected monoclonal antibody are isolated by RT-PCRfrom hybridoma cells and ligated in-frame to human IgG₁ heavy-chain andkappa light chain constant regions, respectively, in mammalianexpression vectors. Alternatively a human Ig expression vector such asTCAE 5.3 is used that contains the human IgG₁ heavy-chain and kappalight-chain constant region genes on the same plasmid (Preston et al.,1998, Infection & Immunity 66:4137-42). Expression vectors encodingchimeric heavy- and light-chains are then co-transfected into Chinesehamster ovary (CHO) cells for chimeric antibody production.Immunoreactivity and affinity of chimeric antibodies are compared toparental murine antibodies by ELISA and FACS.

Humanized Antibodies

As chimeric antibody therapeutics are still frequently antigenic,producing a human anti-chimeric antibody (HACA) immune response,chimeric antibodies against DDR2 can undergo further humanization. Togenerate humanized antibodies, key aspects of the specificitydetermining motifs of the antibody, potentially including elements fromboth the three short hypervariable sequences, or complementarydetermining regions (CDRs), and/or the framework regions required tocorrectly position the CDR regions of the antibody heavy- andlight-chain variable domains described above are engineered usingrecombinant DNA technology into the germline DNA sequences of humanheavy- and light-chain antibody genes, respectively, and then clonedinto a mammalian expression vector for expression in CHO cells. Theimmunoreactivity and affinity of the humanized antibodies are comparedto parental chimeric antibodies by ELISA and FACS. Additionally,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of the humanized antibody.

Human Antibodies

In some embodiments, human antibodies that specifically recognize theextracellular domain of DDR2 can be isolated using phage displaytechnology. A phage display antibody library containing human antibodyvariable domains displayed as single chain Fv or as fab domains isscreened for specific and high affinity recognition of the DDR2 antigendescribed above. The identified variable domain antibody sequences arethen reformatted into an Ig expression vector containing human IgG₁heavy-chain and kappa light-chain for expression of human antibodies inCHO cells.

Example 2 In Vitro Assays to Evaluate Antibodies Against DDR2

This example describes representative in vitro assays to test theactivity of antibodies generated against DDR2 on cell proliferation,pathway activation, and cytotoxicity.

Complement-Dependent Cytotoxicity Assay

In certain embodiments, cancer cell lines expressing DDR2 or cancer stemcells isolated from a patient sample passaged as a xenograft inimmunocompromised mice (described in detail below) can be used tomeasure complement dependent cytotoxicity (CDC) mediated by an antibodyagainst DDR2. Cells (10⁶ cells/ml) are suspended in 200 ul RPMI 1640culture medium supplemented with antibiotics and 5% FBS. Suspended cellsare mixed with antibodies against DDR2 or control antibodies intriplicate and incubated for 1 to 4 hours at 37° C. in 5% CO₂. Treatedcells are then collected, resuspended in 100 μl FITC-labeled annexin Vdiluted in culture medium, and incubated at room temperature for 10minutes. One hundred microliters of propidium iodide solution (25 μg/ml)diluted in HBSS is added and incubated for 5 minutes at roomtemperature. Cells are collected, resuspended in culture medium, andanalyzed by flow cytometry. Flow cytometry of FITC stained cellsprovides total cell counts, and propidium iodide uptake by dead cells asa percentage of total cell numbers is used to measure cell death in thepresence antibodies against DDR2 compared to control antibodies. Theability of anti-DDR2 antibodies to mediate complement-dependentcytotoxicity is thus determined.

Antibody-Dependent Cellular Cytotoxicity Assay

In certain embodiments, cancer cell lines expressing DDR2 or cancer stemcells isolated from a patients sample passaged as a xenograft inimmunocompromised mice (described in detail below) can be used tomeasure antibody dependent cellular cytotoxicity (ADCC) mediated by anantibody against DDR2. Cells (10⁶ cells/ml) are suspended in 200 μlphenol red-free RPMI 1640 culture medium supplemented with antibioticsand 5% FBS. Peripheral blood mononuclear cells (PBMCs) are isolated fromheparinized peripheral blood by Ficoll-Paque density gradientcentrifugation for use as effector cells. Target cells (T) are thenmixed with PBMC effector cells (E) at E/T ratios of 25:1, 10:1, and 5:1in 96-well plates in the presence of DDR2 antibody or a controlantibody. Controls include incubation of target cells alone and effectorcells alone in the presence of antibody. Cell mixtures are incubated for1 to 6 hours at 37° C. in 5% CO₂. Released lactate dehydrogenase (LDH),a stable cytosolic enzyme released upon cell lysis, is then measured bya colorimetric assay (CytoTox96 Non-radioactive Cytotoxicity Assay;Promega; Madison, Wis.). Absorbance data at 490 nm are collected with astandard 96-well plate reader and background corrected. The percentageof specific cytotoxicity is calculated according to the formula: %cytotoxicity=100×(experimental LDH release−effector spontaneous LDHrelease−target spontaneous LDH release)/(target maximal LDHrelease−target spontaneous LDH release). The ability of antibodiesagainst DDR2 receptor to mediated antibody dependent cellularcytotoxicity is thus determined.

Example 3 In Vivo Prevention of Tumor Growth Using Anti-DDR2 Antibodies

This example describes the use of anti-DDR2 antibodies to prevent tumorgrowth in a xenograft model. Tumor cells from a patient sample (solidtumor biopsy or pleural effusion) that had been passaged as a xenograftin mice were prepared for repassaging into experimental animals. Tumortissue was removed under sterile conditions, cut up into small pieces,minced completely using sterile blades, and single cell suspensions wereobtained by enzymatic digestion and mechanical disruption. Specifically,pleural effusion cells or the resulting tumor pieces were mixed withultra-pure collagenase III in culture medium (200-250 units ofcollagenase per mL) and incubated at 37° C. for 3-4 hours with pipettingup and down through a 10-mL pipette every 15-20 minutes. Digested cellswere filtered through a 45 μM nylon mesh, washed with RPMI/20% FBS, andwashed twice with HBSS. Dissociated tumor cells were then injectedsubcutaneously into the mammary fat pads of NOD/SCID mice to elicittumor growth.

Dissociated tumor cells were first sorted into tumorigenic andnon-tumorigenic cells based on cell surface markers before injectioninto experimental animals. Specifically, tumor cells dissociated asdescribed above were washed twice with Hepes buffered saline solution(HBSS) containing 2% heat-inactivated calf serum (HICS) and resuspendedat 10⁶ cells per 100 μl. Antibodies were added and the cells incubatedfor 20 minutes on ice followed by two washes with HBSS/2% HICS.Antibodies included anti-ESA (Biomeda, Foster City, Calif.), anti-CD44,anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16, -CD18,-CD31, -CD64, and -CD140b (collectively referred to as Lin; PharMingen,San Jose, Calif.). Antibodies were directly conjugated to fluorochromesto positively or negatively select cells expressing these markers. Mousecells were eliminated by selecting against H2Kd+ cells, and dead cellswere eliminated by using the viability dye 7AAD. Flow cytometry wasperformed on a FACSVantage (Becton Dickinson, Franklin Lakes, N.J.).Side scatter and forward scatter profiles were used to eliminate cellclumps. Isolated ESA+, CD44+, CD24−/low, Lin− tumorigenic cells werethen injected subcutaneously into NOD/SCID mice to elicit tumor growth.

Anti-DDR2 antibodies were analyzed for their ability to reduce thegrowth of PE13 breast tumor cells. Dissociated PE13 cells (10,000 peranimal) were injected subcutaneously into the flank region of 6-8 weekold NOD/SCID mice (n=10). Two days after tumor cell injection, animalswere injected intraperitoneal (i.p.) with 10 mg/kg anti-DDR2 antibodiestwo times per week. Tumor growth was monitored weekly until growth wasdetected, after which point tumor growth was measured twice weekly.Treatment of animals with anti-DDR2 antibody 9M58 significantly reducedtumor growth as compared to PBS injected controls as well as 9M57anti-DDR2 antibodies (FIG. 2).

Anti-DDR2 antibodies were analyzed for their ability to reduce thegrowth of C9 colon tumor cells. Dissociated C9 cells (10,000 per animal)were injected subcutaneously into the flank region of 6-8 week oldNOD/SCID mice (n=10). Two days after tumor cell injection, animals wereinjected intraperitoneal (i.p.) with 10 mg/kg anti-DDR2 antibodies twotimes per week. Tumor growth was monitored weekly until growth wasdetected, after which point tumor growth was measured twice weekly.Treatment of animals with anti-DDR2 antibody 9M58 significantly reducedtumor growth as compared to PBS injected controls (FIG. 3).

Example 4 In Vivo Prevention of Tumor Growth Using Anti-DDR2 Antibodiesin Combination Therapy

This example describes the use of anti-DDR2 antibodies to prevent tumorgrowth in a xenograft model in combination with chemotherapy. Tumorcells from a patient solid tumor biopsy passaged as a xenograft in micewere prepared for repassaging into experimental animals as described indetail above.

In certain embodiments, anti-DDR2 antibodies were analyzed for theirability to affect breast tumor recurrence after combination therapy witha chemotherapeutic. Dissociated PE13 cells (10,000 per animal) wereinjected subcutaneously into the flank region of 6-8 week old NOD/SCIDmice (n=10) and animals are monitored for tumor growth. Once tumorsreached an average size of approximately 100 mm³, treatment began:animals were treated i.p. with 10 mg/kg 9M58 anti-DDR2 antibodies orcontrol antibodies in combination with 15 mg/kg taxol administered twotimes per week for a total of four weeks. Tumor growth was monitoredweekly as tumor volume decreased in response to the combination therapy.After four weeks, taxol administration ceased while antibody treatmentwas maintained. Continual treatment with anti-DDR2 antibody 9M58significantly reduced tumor reoccurrence after combination therapy ascompared control antibodies (FIG. 5).

Example 5 In Vivo Treatment of Tumors Using Anti-DDR2 Antibodies

This example describes the use of anti-DDR2 antibodies to treat cancerin a xenograft model. In certain embodiments, tumor cells from a patientsample (solid tumor biopsy or pleural effusion) that have been passagedas a xenograft in mice can be prepared for repassaging into experimentalanimals. Tumor tissue is removed, cut up into small pieces, mincedcompletely using sterile blades, and single cell suspensions obtained byenzymatic digestion and mechanical disruption. Dissociated tumor cellsare then injected subcutaneously either into the mammary fat pads, forbreast tumors, or into the flank, for non-breast tumors, of NOD/SCIDmice to elicit tumor growth. Alternatively, ESA+, CD44+, CD24−/low, Lin−tumorigenic tumor cells are isolated as described in detail above andinjected.

Following tumor cell injection, animals are monitored for tumor growth.Once tumors reach an average size of approximately 150 to 200 mm³,antibody treatment begins. Each animal receives 100 μg of DDR2antibodies or control antibodies i.p. two to five times per week for atotal of 6 weeks. Tumor size is assessed twice a week during these 6weeks. The ability of DDR2 receptor antibodies to prevent further tumorgrowth or to reduce tumor size compared to control antibodies is thusdetermined.

At the end point of antibody treatment, tumors are harvested for furtheranalysis. In some embodiments a portion of the tumor is analyzed byimmunofluorescence to assess antibody penetration into the tumor andtumor response. A portion of each harvested tumor from anti-DDR2 treatedand control antibody treated mice is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 μm sections onto glassslides. In some embodiments, a portion of each tumor is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 μm section onto glassslides. Sections are post-fixed and incubated with chromophore labeledantibodies that specifically recognize injected antibodies to detectanti-DDR2 or control antibodies present in the tumor biopsy. Furthermoreantibodies that detect different tumor and tumor-recruited cell typessuch as, for example, anti-VE cadherin (CD144) or anti-PECAM-1 (CD31)antibodies to detect vascular endothelial cells, anti-smooth musclealpha-actin antibodies to detect vascular smooth muscle cells, anti-Ki67antibodies to detect proliferating cells, TUNEL assays (Negoescu et al.,E. J Histochem Cytochem. 1996 September; 44(9):959-68; Negoescu et al.,Biomed Pharmacother. 1998; 52(6):252-8)) to detect dying cells,anti-β-catenin antibodies to detect Wnt signaling, andanti-intracellular domain (ICD) Notch fragment antibodies to detectNotch signaling can be used to assess the effects of antibody treatmenton, for example, angiogenesis, tumor growth, and tumor morphology.

In certain embodiments, the effect of anti-DDR2 antibody treatment ontumor cell gene expression is also assessed. Total RNA is extracted froma portion of each harvested tumor from DDR2 antibody treated and controlantibody treated mice and used for quantitative RT-PCR. Expressionlevels of DDR2 as well as addition cancer stem cell markers previouslyidentified (e.g. CD44) are analyzed relative to the house-keeping geneGAPDH as an internal control. Changes in tumor cell gene expression uponDDR2 antibody treatment are thus determined.

In addition, the effect of anti-DDR2 antibody treatment on the presenceof cancer stem cells in a tumor is assessed. Tumor samples from DDR2versus control antibody treated mice are cut up into small pieces,minced completely using sterile blades, and single cell suspensionsobtained by enzymatic digestion and mechanical disruption. Dissociatedtumor cells are then analyzed by FACS analysis for the presence oftumorigenic cancer stem cells based on ESA+, CD44+, CD24−/low, Lin−surface cell marker expression as described in detail above.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin− expression following anti-DDR2 antibody treatment can thenassessed. ESA+, CD44+, CD24−/low, Lin− cancer stem cells isolated fromDDR2 antibody treated versus control antibody treated mice arere-injected subcutaneously into the mammary fat pads of NOD/SCID mice.The tumorigenicity of cancer stem cells based on the number of injectedcells required for consistent tumor formation is then determined.

Example 6 Prevention of Metastasis with Anti-DDR2 Antibodies

This example describes methods of treating metastasis using antibodiesagainst DDR2. The role of DDR2 in regulation of the extracellular matrixis well-established and suggests that antibodies directed against DDR2may inhibit invasion and metastasis of tumor cells. Primary tumor cellsor cells from a primary tumor cell line can be transplanted intoNOD/SCID mice as described, for example, in Wang et al., 2005, Lab.Investigation 85: 1395-1404. In certain embodiments, antibody treatmentcommences following detection of tumor growth of transplanted cellswithin the original transplant site. Injection of 10 mg/kg anti-DDR2antibodies intraperitoneal (i.p.) two times per week continues for up to54 weeks. The spread of tumor cells to organ systems beyond the originaltransplant site is monitored compared to PBS injected control animals.

Example 7 Treatment of Human Cancer Using Anti-DDR2 Antibodies

This example describes methods for treating cancer using antibodiesagainst DDR2 to target tumors comprising cancer stem cells and/or tumorcells in which DDR2 expression has been detected. The presence of cancerstem cell marker expression can first be determined from a tumor biopsy.Tumor cells from a biopsy from a patient diagnosed with cancer areremoved under sterile conditions. In some embodiments the tissue biopsyis fresh-frozen in liquid nitrogen, embedded in O.C.T., and cut on acryostat as 10 μm sections onto glass slides. In some embodiments, thetissue biopsy is formalin-fixed, paraffin-embedded, and cut on amicrotome as 10 μm section onto glass slides. Sections are incubatedwith antibodies against DDR2 to detect protein expression.

The presence of cancer stem cells can also be determined. Tissue biopsysamples are cut up into small pieces, minced completely using sterileblades, and cells subject to enzymatic digestion and mechanicaldisruption to obtain a single cell suspension. Dissociated tumor cellsare then incubated with anti-ESA, -CD44, -CD24, -Lin, and -DDR2antibodies to detect cancer stem cells, and the presence of ESA+, CD44+,CD24−/low, Lin−, DDR2+ tumor stem cells is determined by flow cytometryas described in detail above.

Cancer patients whose tumors are diagnosed as expressing DDR2 aretreated with anti-DDR2 antibodies. In certain embodiments, humanized orhuman monoclonal anti-DDR2 antibodies generated as described above arepurified and formulated with a suitable pharmaceutical vehicle forinjection. In some embodiments, patients are treated with the DDR2antibodies at least once a month for at least 10 weeks. In someembodiments, patients are treated with the DDR2 antibodies at least oncea week for at least about 14 weeks. Each administration of the antibodyshould be a pharmaceutically effective dose. In some embodiments,between about 2 to about 100 mg/ml of an anti-DDR2 antibody isadministered. In some embodiments, between about 5 to about 40 mg/ml ofan anti-DDR2 antibody is administered. The antibody can be administeredprior to, concurrently with, or after standard radiotherapy regimens orchemotherapy regimens using one or more chemotherapeutic agent, such asoxaliplatin, fluorouracil, leucovorin, or streptozocin. Patients aremonitored to determine whether such treatment has resulted in ananti-tumor response, for example, based on tumor regression, reductionin the incidences of new tumors, lower tumor antigen expression,decreased numbers of cancer stem cells, or other means of evaluatingdisease prognosis.

Example 8 Cancer Stem Cells Express Differential Levels DDR2

To look at the expression DDR2 in tumorgenic cancer stem cells versusnon-tumorgenic tumor cells, DDR2 gene expression in the two cellpopulations was compared by microarray analysis. Dissociated PE13 breasttumor cells were sorted by FACS into ESA+CD44+CD24−/lowLin− tumorigeniccells and non-tumorigenic cells as described in detail above. Probeswere prepared from mRNA isolated from the two cell populations andhybridized to Affymetrix HG-U133 gene chips using standard experimentalprotocols. A comparison of relative gene expression revealed that cancerstem cells express elevated levels of DDR2 compared to nontumorigeniccells (FIG. 4A).

Similarly, colon tumor cells from two different primary tumors, UMC4 andUMC6, were analyzed for differential expression of DDR2 by tumorigeniccompared to non-tumorigenic cells. Dissociated C4 and C6 cells weresorted by FACS into ESA+CD44+CD24−/lowLin− tumorigenic cells andnon-tumorigenic cells as described in detail above. Probes were preparedfrom mRNA isolated from the two cell populations and hybridized toAffymetrix HG-U133 gene chips. The analysis revealed that cancer stemcells from different colon tumors express different relative expressionlevels of DDR2 compared to non-tumorigenic cells. Specifically, C6 coloncancer stem cells express elevated levels of DDR2 compared tonontumorigenic cells, whereas C4 colon cancer stem cells express lowerlevels of DDR2 compared to nontumorigenic cells (FIG. 4B).

Example 9 Differential Gene Expression in Breast Tumors FollowingTreatment with Anti-DDR2 Antibodies

To look at changes in gene expression in tumor cells after treatmentwith anti-DDR2 antibodies, microarray analysis was conducted on treatedversus control treated tumors. PE13 breast tumor xenografts in NOD/SCIDmice were treated with anti-DDR2 antibodies (9M58) or PBS, and then ESA+tumor cells were isolated as described in detail above. Probes wereprepared from mRNA isolated from three anti-DDR2 antibody treated tumorsand three control tumors and hybridized to Affymetrix HG-U133 gene chipsusing standard experimental protocols. A comparison of relative geneexpression revealed a number of genes differentially expressed followingtreatment with anti-DDR2 antibodies (Table 1).

TABLE 1 Gene Expression Changes in Response to anti-DDR2 AntibodyTreatment Down-Regulated Genes Up-Regulated Genes EGFR NOTCH2 CYR61 SOS1ATF3 TCF7L2 MAFF CD44 NR4A3 NUMB BTG2 CDK6 DUSP1 SMYD2 RHOB SOX1 NFKBIA

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

All documents, e.g., scientific publications, patents, patentapplications and patent publications, recited herein are herebyincorporated by reference in their entirety to the same extent as ifeach individual document was specifically and individually indicated tobe incorporated by reference in its entirety. Where the document citedonly provides the first page of the document, the entire document isintended, including the remaining pages of the document.

SEQ ID NO: 1 Human DDR2 1-399MILIPRMLLVLFLLLPILSSAKAQVNPAICRYPLGMSGGQIPDEDITASSQWSESTAAKYGRLDSEEGDGAWCPEIPVEPDDLKEFLQIDLHTLHFITLVGTQGRHAGGHGIEFAPMYKINYSRDGTRWISWRNRHGKQVLDGNSNPYDIFLKDLEPPIVARFVRFIPVTDHSMNVCMRVELYGCVWLDGLVSYNAPAGQQFVLPGGSIIYLNDSVYDGAVGYSMTEGLGQLTDGVSGLDDFTQTHEYHVWPGYDYVGWRNESATNGYIEIMFEFDRIRNFTTMKVHCNNMFAKGVKIFKEVQCYFRSEASEWEPNAISFPLVLDDVNPSARFVTVPLHHRMASAIKCQYHFADTWMMFSEITFQSDAAMYNNSEALPTSPMAPTTYDPMLKVDDSNTR

1-34. (canceled)
 35. A method of treating cancer, the method comprising:administering to a patient with a solid tumor a therapeuticallyeffective amount of a monoclonal antibody that specifically binds humanDDR2 within a region comprising amino acids 24 to
 241. 36. The method ofclaim 35, wherein the antibody binds the discoidin/coagulation domain ofhuman DDR2.
 37. The method of claim 35, wherein the antibody is achimeric antibody.
 38. The method of claim 35, wherein the antibody is ahumanized antibody.
 39. The method of claim 35, wherein the antibody isa human antibody.
 40. The method of claim 35, wherein the antibody isantibody 9M58.
 41. The method of claim 35, wherein the antibody isconjugated to a cytotoxic moiety.
 42. The method of claim 35, furthercomprising administering at least one additional therapeutic agent tothe patient.
 43. The method of claim 35, wherein the antibody isadministered in a pharmaceutical composition.
 44. The method of claim35, wherein the solid tumor is selected from the group consisting of abreast tumor, colorectal tumor, lung tumor, pancreatic tumor, prostatetumor, and a head and neck tumor.
 45. A method of treating cancer, themethod comprising: administering to a patient with a solid tumor atherapeutically effective amount of a monoclonal antibody that competesfor specific binding to human DDR2 with the antibody 9M58 which isproduced by the hybridoma deposited with ATCC having deposit numberPTA-10415.
 46. The method of claim 45, wherein the antibody competes forspecific binding within the region comprising amino acids 24 to 241 ofhuman DDR2.
 47. The method of claim 45, wherein the antibody competesfor specific binding to the discoidin/coagulation domain of human DDR2.48. The method of claim 45, wherein the antibody is a chimeric antibody.49. The method of claim 45, wherein the antibody is a humanizedantibody.
 50. The method of claim 45, wherein the antibody is a humanantibody.
 51. The method of claim 45, wherein the antibody is conjugatedto a cytotoxic moiety.
 52. The method of claim 45, further comprisingadministering at least one additional therapeutic agent to the patient.53. The method of claim 52, wherein the additional therapeutic agent isa chemotherapeutic agent.
 54. The method of claim 45, wherein theantibody is administered in a pharmaceutical composition.
 55. The methodof claim 45, wherein the solid tumor is selected from the groupconsisting of a breast tumor, colorectal tumor, lung tumor, pancreatictumor, prostate tumor, and a head and neck tumor.